Studies Of Polyamides That Are Active Antiviral Agents Against HPV16

132 Pages·2017·3.58 MB·English

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9-5-2016
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Edith Csiki-Fejer
University of Missouri-St. Louis, [email protected]
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IN VIVO AND IN VITRO STUDIES OF POLYAMIDES THAT ARE ACTIVE ANTIVIRAL
AGENTS AGAINST HPV16
by
Edith Csiki-Fejer
M.S. in Chemistry / Biochemistry, May, 2014, University of Missouri-St. Louis
M.S. in Chemistry / Physical, June, 1996, University of Transylvania-Brasov

A Thesis
Submitted to The Graduate School of the
University of Missouri-St. Louis
in partial fulfillment of the requirements for the degree
Doctor of Philosophy
In
Chemistry
With an Emphasis in Biochemistry
August 2016

Advisory Committee

James K. Bashkin, Ph.D.
Chairperson
Benjamin J. Bythell, Ph.D.
Keith J. Stine, Ph.D.
Chung F. Wong, Ph.D.

i
For
Ágota, Mátyás, Anna,
and Bálint

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Acknowledgements
It has been a privilege to work under the direction of Prof. James K. Bashkin at
UMSL. Under his supervision, I had the freedom and support to be able to pursue my
ideas. Working in the Bashkin group over the last five years has allowed me to learn many
research techniques and collaborate with a large group of scientists. I would like to express
my appreciation for his guidance. I am tremendously thankful for his patience and
assistance.
I am also grateful to the supportive faculty at UMSL, a distinguished group of
scientists, and to the members of my thesis committee who have always been available
for advice or assistance. I will always be grateful to Prof. Cynthia M. Dupureur for her
useful insights on footprinting experiments and Prof. Michael E. Hughes and his assistant
Erin Arant for their training on next generation sequencing and stimulating scientific
discussions.
Among the former members of the Bashkin group, I’m glad to have known Gaofei
He, former post-doc, Priyanka Bapat, Faten Tamimi, and Silke Evdokimov, great
colleagues who made tough days bearable. I was fortunate to have worked with Fanny
Hakami on the bioavailability project and enjoy her uplifting spirit, which helped me through
my first years of graduate school. The precursory work to my projects, the synthesis and
purification of the polyamides, was done by Dr. Kevin Koeller and Dr. George Harris. I
must also thank Dr. E. Vasilieva, J. Niederschulte, Y. Song (members of Dupureur lab),
and Dr. Shana Terrill and Astha Ahuja, members of the Nichols Lab, for introducing me to
cell culture work. Among current members of the group, thanks to Jose’ Scuderi and
Carlos Castañeda, who always were helpful lab mates during four years of intensive work.
Our discussions helped shape the direction of the work presented herein.
I would also like to acknowledge our collaborators at NanoVir, LLC, Dr. T.G.
Edwards and Dr. C. Fisher, for their antiviral potency studies. Special thanks to our
collaborators at the Univ. of Missouri, Colombia, the DNA Core Facility members
(especially E. Kessler) who ran numerous samples prepared for the CE, as well as N.
Bivens, who performed the sequencing on a HiSeq 2500 Illumina instrument. Also, I am
eagerly looking forward to the sequencing results’ interpretation that is being performed
at Informatics Research Core Facility by Christopher Bottoms under the supervision of Dr.
Scott Givan.
Many thanks to my family, the most important people in my life. Without their
mutual understanding and support this work would not have been completed.
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Abstract
Long-term, persistent infection with high-risk strains of human papillomavirus (HPV)
is the precursor of most cervical cancers and an increasing number of the head and neck
cancers. While HPV vaccines can protect patients under twenty-five years old from
possible infections, no HPV antiviral drugs are available for the definitive treatment of
preexisting or future viral infections. To treat current or future HPV infections, drugs that
selectively block virus-specific processes, but do not damage the host cells, are needed.
The compounds developed in our group are unique Imidazole-pyrrole polyamides,
analogs of natural products Distamycin A and Netropsin that function by interfering with
natural virus-host interactions.
Within the overall program of my group, my work focuses on three specific projects.
The first project (Chapter 2) includes bioavailability LC-MS/MS analysis study of one of
the leading compounds in plasma and whole blood.
The second project includes the biophysical study of interactions between two
polyamides, called TMG Asymmetric Hairpin Polyamides (TMG-AHP), and an essential
viral DNA segment. The aim of this study was to determine where and how strongly the
compounds bind to the viral DNA. In this work, two methods were used: the quantitative
deoxyribonuclease (DNase I) footprinting method and the affinity cleavage assay (AC).
The results are presented in Chapters 3 and 4. The remarkable findings resulted in binding
location maps that help us to better understand the mechanism of action of AHPs and to
address the question: what is the primary reason for neutralizing a virus by our
polyamides.
Based on the knowledge gathered from the results of the second project, a complex
study (RNA-Seq) concerned with genome expression was performed. The aim of the third
project is to find the common features and differences in the polyamides’ mechanism of
action by quantifying messenger RNA (mRNA), after treatment of HPV infected skin cells
with 8 different polyamides.
In parallel with the analysis of human and viral transcriptome, a separate study
concerned with the differential expression profile of seven DDR genes that are
components of the homologous recombination (HR) pathway have been studied by RT-
qPCR method. The results are presented in Chapter 5 and 6. Data gathered from the third
project helps us further understand the mechanism of polyamide action.

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Table of Contents
Page
ACKNOWLEDGEMENTS………………….…………..…...………………………….I
ABSTRACT…………………………………….………….……………………………IV
LIST OF FIGURES……………………………………………..………………….…...V

Chapter 1
Introduction

Figure 1.1 Episome copy numbers in different phases of replication…………………...…6
Figure 1.2 Model of E2-mediated Tethering of the Viral Genome to
Host Chromatin……………………………………………………………….............................7
Figure 1.3 The HPV circular dsDNA genome structure…………………………………......8
Figure 1.4 DNA damage response to ss and ds breaks…………………………………….9

Table 1.1 Polyamides used for RNASeq studies……………………………......…….…...14
Table 1.2 Sequences of polyamides (PAs). PA1 and PA25 are the
original preclinical lead compounds……………………………………………………….…..14

Chapter 2
Polyamide Bioavailability

Figure 2.1 Structure of NV1042……………………………………………………………....17
Figure 2.2 Structure of NV1057………………………………………………………………17
Figure 2.3 Representative mass spectra of NV1042 precursor ions………………...….....21
Figure 2.4 The calibration curve …………………………………………………………......22
Figure 2.5 Representative chromatograms of NV1042…………………………………….24

Table 2.1 Positive electrospray ionization of the NV1057…………………………….....….17
Table 2.2 HPLC peak areas obtained following the pellet sample
preparation procedure………………………………………………………………………......22

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Chapter 3
Binding Studies of TMG Asymmetric Hairpin Polyamides (TMG-AHP)
NV1078 and NV1087 with Designed, 120 bp DNA Sequences

Figure 3.1 Fragments generated in the DNase I reactions and
Sanger Sequencing…………………………………………………………………....27
Figure 3.2 5’-end labeled fragments from affinity cleavage and
Maxam-Gilbert sequencing…………………………………………………..…….…28
Figure 3.3 Example of Sanger (USB) sequencing electropherogram
of 120XC in FAM channel………………………………………………………. …..30
Figure 3.4 The structure of hairpin TMG NV1078 polyamide…………………….32
Figure 3.5 The structure of hairpin TMG NV1087 polyamide………………….....32
Figure 3.6 Electropherograms of 120 XC bp DNA..…………………………….....33
Figure 3.7 Isotherm (KaleidaGraph 4.1 software)..……………………………......34
Figure 3.8 Dissociation constants for the binding sites on 120 bp
DNA of NV107…………………………………………………………………….…...36
Figure 3.9 Dissociation constants for the binding sites on 120 bp
DNA of NV1087…………………………………………………………………….…..37
Figure 3.10 Binding of NV1087 the position with systematic changes…………...37
Figure 3.11 Isotherm triplicates of 3 binding sites of NV1078……………………..39
Figure 3.12 Isotherm triplicates of 4 binding sites of NV1087……………….…....41

Table 3.1 Primer pairs that extend the 90 bp DNA to 120 bp DNA…………....….26
Table 3.2 Summary of Hill dissociation constants NV1078…………………….…35
Table 3.3 Summary of Hill association constants NV1078………………………..34
Table 3.4 Summary of Hill dissociation constants NV1087………………….…….36

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Chapter 4
Interactions Between two TMG Asymmetric Hairpin Polyamides
(TMG-AHP), NV1078 and NV1087, with the HPV16,18 LCR segment

Figure 4.1 The structure of hairpin TMG NV1078 polyamide and the code of DNA
recognition…………………………………………………………...…………………47
Figure 4.2 The structure of hairpin TMG NV1087 polyamide and the code of
DNA recognition…………………………………………………………………….….47
Figure 4.3 Binding sites of NV1078 on 365 bp LCR HPV16 fragment…….…........49
Figure 4.4 Binding affinity of NV1078 on HPV16 LCR sequence………….……....54
Figure 4.5 Binding sites of NV1087 on 365 bp LCR HPV16 fragment………….....55
Figure 4.6 Binding affinities of NV1087 on HPV16 LCR sequence……………......58
Figure 4.7 DNA binding sequence for forward binding of NV1087 on LCR…….....59
Figure 4.8 DNA binding sequence for reverse binding of NV1087 on LCR…….....59
Figure 4.9 Sample electropherogram illustrating the enhanced affinity
cleavage sites of NV1078 on LCR HPV16 and LCR HPV18…………………….…60
Figure 4.10 Hydrogen bonds formed by NV1078 polyamide………………...…….61
Figure 4.11 Comparison of NV1078 and NV1087 AC sites on LCR……….……...64
Table 4.1 Binding sites of TMG-AHP, NV1078 on LCR…………………………….51
Table 4.2 The most probable binding sites of NV1078 to LCR…………...……..…53
Table 4. 3 Binding sites of TMG-AHP, NV1087 on HPV16…………………………56
Table 4.4 The most probable binding sequences of NV1087 on LCR………….…57

Chapter 5
Differential DDR Gene Expression Profile of W12E Cells After
Treatment with Active Anti-HPV Hairpin Polyamides

Figure 5.1 Mitomycin C structure………………………………………………..…...73
Figure 5.2 The Keratinocyte W12E cells and 3T3 image……………………..…..74
Figure 5.1 (A) SYBR Green I structure, (B) SYBER Green I upon binding to
dsDNA……………………………………………………………………………..…....80
Figure 5.4 ROX chemical formula…………………………………………….…......80
Figure 5.5 Downregulation of DDR genes in cells treated with polyamides.
C-1 Series………………………………………………………………………….......86
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Figure 5.6 Downregulation of DDR genes in cells treated with polyamides.
C-2 Series……………………………………………………………………….…......87
Figure 5.7 Downregulation of DDR genes in cells treated with polyamides.
C-3 Series………………………………………………………………………....……88
Figure 5.8 Downregulation of DDR genes in cells treated with polyamides.
C-4 Series………………………………………………………………………...…….89
Figure 5.9 Downregulation of DDR genes in cells treated with polyamides.
C-5 Series………………………………………………………………………...…….90
Figure 5.10 Gene expression downregulation induced by NV1078,
NV1042 and NV1011………………………………………………………………….92

Table 5. 1 Viral proteins that activate the DDR pathways…………………….......69
Table 5. 2 Polyamides used in RT-qPCR studies…………………………..……...72
Table 5. 3 The W12E and the 3T3 passage numbers used for obtaining
5 series of biological replicates………………………………………..……………..74
Table 5. 4 RIN, volumes and concentrations of total RNA samples used for
reverse transcription corresponding to 1 µg sample…………………….……..….78
Table 5.5. Target DDR genes used in the study…………………………….….….81
Table 5.6. Reference genes level of expression and function……………….…...82
Table 5.7. Cq values of qPCR experiments of C-
Series……………………………………………………………................................86
Table 5.8. Reference genes stability values associated with qPCR
experiments of C-1 Series……………………………………………………...…...86
Table 5.9 Cq values of qPCR experiments of C-2 Series ……………….……….87
Table 5.10. Reference genes stability values associated with qPCR
experiments of C-2 Series……………………………………………………….......87
Table 5.11. Cq values of qPCR experiments of C-3 Series……………...…...…..88
Table 5.12. Reference genes stability values associated with qPCR
experiments of C-1 Series………………………………………………...…………88
Table 5.13. Cq values of qPCR experiments of C-4 Series……………………...89
Table 5.14. Reference genes stability values associated with qPCR
experiments of C-4 Series……………………………………………………...…....89
Table 5.15. Cq values of qPCR experiments of C-5 Series……………………...90
Table 5.16. Reference genes stability values associated with qPCR
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experiments of C-5 Series…………………………………………………....……...90
Table 5.17. The IC50 and IC90 values and their relationships to 0.1 µM
concentration…………………………………………………………………………...91
Table 5.18. Comparison of gene expression altered by NV1042
(PA25) in W12E cells, induced in two different conditions………………...…...…94
Table 5.19. Nine pair-wise (3 DMSO treated ΔCq values
x 3 PA treated ΔCq values) comparisons……………………………………….….95

Chapter 6
The Analysis of Viral and Human Transcriptomes After Treatment of
W12E Cells with Polyamides

Figure 6.1 The HPV genome circular dsDNA structure…………………...…...….99
Figure 6.2 TruSeq Stranded mRNA Library quality profiles
generated on Agilent 2100 BioAnalyzer…………………………………………...105

Table 6.1 Polyamides used for RNASeq studies…………………………...…...…99
Table 6.2. Adapters associated with different samples…………………......…...101
Table 6.3. TruSeq Stranded mRNA Libraries characteristics…………………...103

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